摘要
近年来,中国大肠癌的发病率以及死亡人数呈不断上升趋势,我国的上海、广东等发达地区,每年大肠癌发病人数根式以4.2%速度在增长,其增幅超过西方发达国家的水平。大肠癌因其早期症状不明显,在已确诊患者中中晚期占大多数,是一种恶性程度较高,对人民威胁程度较大的消化道肿瘤,死亡率位居西方发达国家恶性肿瘤死亡率的第2、3位,在发展中国家发病率也逐年升高。据调查显示,目前大肠癌在中国大部分地区已经成为发病率上升最快的恶性肿瘤之一。2002年以后上海市大肠癌发病率已经超越胃癌跃居第2位,仅次于肺癌。当前,目前治疗大肠癌的方法主要是以手术切除为主,辅助以化疗、放疗或免疫治疗,以及中药治疗等。由于大肠癌早期发现率较低,大肠癌术后5年内的复发率、死亡率仍然保存在一个较高的水平。早期发现,有效进疗大肠癌成为的目前医疗及科研工作者的共同目标,如何寻找作为大肠癌早期诊断的新型敏感肿瘤标志物及新的大肠癌治疗靶目标基因,成为目前大肠癌基础及临床研究的热点。本研究关注中介体(Mediator, Med)家族的一个重要成员--Med-19。
RNA干扰技术就是将与内源mRNA编码区同源双链RNA(double-stranded RNA, dsRNA)导入细胞,通过一系列细胞反应引起特异性地降解对应的mRNA,从而导致特定基因表达沉默的一种技术。该项技术简单、高效已成为研究基因功能的有效工具。而利用慢病毒作为siRNA的装载工具,可以在细胞内长时间、稳定地生成siRNA,保证了基因抑制的效率和试验。本实验中,我们根据其mRNA序列设计Med-19基因RNAi的有效靶序列,构建表达siRNA慢病毒载体,为进一步研究Med-19基因在大肠癌发生发展中的作用并且进一步探讨其探讨其作用机制奠定基础。
中介体(Mediator, Med),是RNA聚合酶Ⅱ通用转录装置的重要组成部分,其在在真核mRNA合成的活化和阻抑中起着关键作用。1990年kelleher等人在研究酵母RNA聚合酶Ⅱ转录功能时发现,并将其分离,通过最近几年的研究Med复合物的亚基组成及其相关的功能活性已经被逐步揭开。Med的功能包括促进激活的转录、增强基础转录、增强CTD的磷酸化。作为Med家族的重要一员,Med-19在细胞生长中起到重要作用,可直接或间接参与、介导多种信号途径。已有研究发现：Med19基因的表达在高侵袭性肺癌细胞的表达要明显高于低侵袭性细胞。
本实验,首先在组织水平上,我们通过Realtime-PCR以及免疫组化的方法比较检测大肠癌及正常大肠肠壁组织中的Med-19mRNA及蛋白质含量,结果发现大肠癌组织中的该基因mRNA的表达量明显高于正常组织。这样为进一步在细胞水平研究该基因奠定了基础。
其次,我们应用大肠癌细胞进行Med-19基因沉默相关功能研究。第一步制备以慢病毒为载体的Med-19-siRNA,根据Med-19基因信息设计Med-19-siRNA序列,成功构建慢Med-19-siRNA病毒载体载体,通过Western Blot筛选有效靶点,寻找效率最高siRNA,通过易转染的293T细胞转染验证其转染能力。成功构建慢病毒载体后,我们选择大肠癌细胞进行试验,试验主要在大肠癌细胞系DLD-1与RKO上进行。首先应用病毒载体对大肠癌细胞进行转染,荧光免疫试验证实慢病毒载体成功感染了大肠癌基因,感染率超过90%。应用实时定量PCR以及Western Blot方法证实转染Med-19-siRNA大肠癌细胞其Med-19mRNA及蛋白的表达相比空白对照组(正常培养的大肠癌细胞)及阴性对照组(转染空白病毒载体大肠癌细胞)有明显减弱,以上研究结果证明我们设计的Med-19-siRNA成功抑制了大肠癌细胞Med-19基因的表达。
进一步我们进行细胞功能学试验,由以下几个实验组成,MTT试验、Brdu试验、流式细胞试验、克隆形成试验。试验分3组进行：空白对照组、阴性对照组以及基因沉默组。阴性对照组转染慢病毒空白载体,基因沉默组转染Med-19-siRNA。结果发现Med-19基因沉默组大肠癌细胞比较其他两组增殖受到明显抑制,DLD-1与RKO细胞处于繁殖期的比例明显低于正常对照组和阴性对照组,细胞形成群落的数量明显少于对照组。
第三部分的裸鼠成瘤试验,我们选择30支雄性裸鼠分基因沉默组、空白对照组、阴性对照组进行,按组别分别接种相同数量Med-19基因沉默的大肠癌细胞、正常培养的大肠癌细胞、感染空载病毒的大肠癌细胞。于种植后15天、20天、25天分别观察肿瘤体积变化,1月后处死裸鼠后比较肿瘤重量。结果显示：基因沉默组的裸鼠肿瘤体生长明显受到抑制,肿瘤生长速度慢于其他两组,肿瘤的平均体积及重量明显小于其他两组。
综上所述：本研究对大肠癌组织Med-19基因表达进行观察,通过成功制备慢病毒为载体的Med-19-siRNA,对大肠癌细胞Med-19基因进行沉默后发现大肠癌细胞的增殖能力受到明显抑制,成瘤能力减弱。并为更加深入地探讨Med-19基因与大肠癌的发生、发展关系以及Med-19基因参与大肠癌发病的机制提供较好的理论依据。
Colorectal cancer (CRC) is the second most frequent tumor in developed countries,with its growing speed by 4.2% of morbidity and death.in shanghai and guangdong china, which is faster thant western country.The early symptom is so oblivious that many patients may ignorant its threaty,and get the diagnose with a later stage. In Shanghai along 2002, colorectal cancer incidence has gone beyond the first two ranks, second only to lung cancer. Today, the surgical operation is the most important treatment of CRC, supported by chemotherapy, radiotherapy or immunotherapy, as well as traditional Chinese medicine treatment. However, colorectal cancer after 5 years the relapse rate, mortality still remains at a higher level. Finding a positive measures for early detection and effective treatment has become the urgent goal for the medical doctor. So looking for the new tumor markers for early diagnosis of colorectal cancer and new gene for therapeutic targets is a hot spots for basic and clinical research of CRC. This study we are concerned is one important member of Mediator family --- Med-19(mediator complex subunit 19)
The technology of RNA interfere is to import the double-stranded RNA into cell which share the same organ with internal mRNA coding zone, then the degradation of the mRNA happens after a series of reaction which making the special gene silence This teconology simple effective as a tool to reseach the gene. In our experiment we design the target sequnce for Med-19,bulid the lentiviral vectors (LVs),which can generate a long and stable silence effict,and make foundation for the relationship of Med-19 gene and colorectal cancer.
Mediator is an important components of the transcription apparatus for RNA polymeraseⅡin transcription of eukaryotic cells. In 1990 kelleher find it and separate it as an important part of MED famlilay, For these year the function of MED complex has been unveiled.Its function including activing the mRNA tanscription,enhance the basic tanscription.It has been proved that Med-19 has an close relationship with invasive ability.of lung cancer.
Our erperment have 3 succesive parts,For colorectal tissue,we excamine the mRNA and protein of colorectal cancer and nomal colorectal tissue using reverse transcription polymerase chain reaction (RT-PCR) amd immunohistochemisty,The rusults shows that the mRNA and protein of Med-19 in tumor is significant higher than the normal tissue.
After that we conduct the experiment of Med-19 gene silence, we produce the Med-19-siRNA using lentivirus based vectors at first,the Med-19-siRNA is designed by its message,finding the most proper target,test its infectious ability with 293T cell.After that,we choose colorectal cancer cell line DLD-1 and RKO. After being infected we test infectability of cell with fluroimmunoassay,The infect rate of Med-19-siRNA is over 90%.By the useng of PCR and western blot,we prove the mRNA and protein of KD(konck down)group are lower than the control group (normal colorectal cancer cell) and negative control group (colorectal cell with empty lentivirus based vectors),all of this can prove the Med-19-siRNA can suppress the expresion of Med-19 gene.
The cell function experiment including the following parts:MTT,Brdu,Colony formation PI-FACS.The reslts shows that the ability of proliferation is suppressed by the silencing of Med-19 gene.
The xenograft experiment is perfomed on 30 nude mices,which divided into 3 groups., The CON(control) group injected with colorectal cancer cell,and NC(negetive control) group injectied with lentivirus vector without any siRNA,with KD(konck down)group injected with lentivirus vector with Med-19siRNA. We observe the tumor size with lenth and width in day 15 and 20 after injected.Turmor weight was compared after the mice was executed 30 days after injected.The results shows that the growth of xenograft tumor is restrained,the number of cell in reproduction is lower comparatively
Generally speaking, we analysed the expression of Med-19 in colorectal tumor and produce the lentivirus vector with Med-19 siRNA and find that the growth of coloectal cell is restrained by silencing the expression of Med-19 gene.
引文
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